REDUCED FOLATE DERIVATIVES ARE ENDOGENOUS SUBSTRATES FOR CMOAT IN RATS

We examined the role of the canalicular multispecific organic anion tr ansporter (cMOAT) in the biliary excretion of reduced folate derivativ es in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and m utant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hered itarily deficient. In vivo, the biliary excretion of endogenous tetrah ydrofolate (H(4)PteGlu), 5-methyltetrahydrofolate (5-CH3-H(4)PteGlu), and 5,10-methylenetetrahydrofolate (5,10-CH2-H(4)PteGlu) in EHBR was r educed to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H(4)PteGlu) was detected onl y in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, signif icant ATP-dependent uptake of H-3-labeled 5-CH3-H4PteGlu into canalicu lar membrane vesicles was observed only in SDR. This ATP-dependent upt ake was saturable with a Michaelis constant (K-m) value of 126 mu M, w hich was comparable with its inhibitor constant (K-i) value of 121 mu M for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinit rophenyl-S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the upta ke of 5-CH3-H(4)PteGlu with a K-i of 35 mu M, which was similar to its K-m value. In addition, H(4)PteGlu and 5,10-CH2-H(4)PteGlu also inhib ited the ATP-dependent uptake of DNP-SG. These results indicate that 5 -CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefo re, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieti es.